Our overall objective is to develop a long-term, culture system for adult rat left ventricular tissue slices whereby myocyte atrophy is prevented by continually pacing the slices to contract isometrically. This approach will bridge the gap between the adult isolated heart preparation and cell culture allowing for a greater degree of control, long term stability and viability and a relatively undisturbed extracellular environment. We are working on the principle that if the adenylate triphosphate pool (ATP) is preserved throughout the sequential stages of organ excision, tissue slicing, and ventricular slice incubation, we can preserve the viability of the tissue slices for up to 7 days, if not longer. The advantages to such a system include: 1) conditions in which all myocardial cells retain their native three-dimensional structural integrity and intercellular interactions;2) ability to perform multiple perturbations on an individual heart, thereby minimizing biological variability;and 3) relatively long term effects of a perturbation can be investigated under highly controlled conditions. Also, the myocardial response would be solely intrinsic in that, regardless of the intervention, it would be void of reflexive adjustments in ventricular loading conditions, heart rate and contractility and variations in neurohormonal background. Most studies using myocardial tissue slices have been limited to several hours of data collection with no attempt to ensure thin, uniform slices, to optimize culture conditions and to stimulate contractions. In order to extend the viability of the tissue slice, our strategy is to establish optimal conditions for viability in a stepwise fashion. To prevent myocyte atrophy, we will test two prototype systems which are designed to impose a physiologic myocardial stretch (preload) and to stimulate the slice to contract isometrically. We will evaluate tissue slice functional viability along with assays for gene expression, apoptosis, ischemic damage (histology or biochemical assays for ATP and lactate dehydrogenase), myocyte atrophy (myocyte cell size), and myocyte injury (release of creatine kinase-MB into tissue slice culture media). There are unlimited applications associated with the proposed system that would be relevant to each cardiac cell type and their interactions with each other and the extracellular environment. Clearly, such a preparation has tremendous potential to contribute significantly to our understanding of mechanisms at the molecular, cellular and tissue levels in normal, genetically modified, aged, male and female, and diseased hearts. Thus, while this is a high- risk undertaking, it would become a high-reward endeavor if successful. PUBLIC HEALTH RELEVANCE: The cardiac remodeling that occurs in response to hypertension, mitral valve regurgitation or heart muscle injury will eventually lead to heart failure. There is a critical need for a cardiac tissue slice culture system that remains alive for up to 7 days in order to investigate at the molecular, cellular and tissue levels the mechanisms that cause the heart to fail. To develop such a system is the objective of this project.